The quantification of nucleic acids plays an important role in the fields of biology and medicine. For example, quantification of nucleic acid is important in cancer diagnosis and prognosis and viral diagnosis and judgments of therapeutic effects (e.g., for HCV and HIV). HCV RNA quantification is important for patients taking IFN. The effect of IFN therapy can be directly found by monitoring the amount of virus during IFN therapy. This enables more effective IFN therapy that is tailored to clinical conditions of each patient. Quantification of target nucleic acid is important for diagnosis of diseases in the future. For example, earlier diagnosis can be effected by examining the expression level of mRNA that responds to exogenous stimuli in the case of a disease that results from exogenous stimuli.
The polymerase chain reaction can be employed for nucleic acid quantification. However, when PCR is employed, the absolute amount of the amplified nucleic acids does not accurately reflect the amount of the target nucleic acid that had existed when amplification was initiated. At first, the amount of the product amplified by PCR generally exponentially increases every cycle, however, the rate of increase slows down and then stops when the amount of the amplified product exceeds a certain level. Thus, the final amount of the amplified product is constant regardless of the amount of the target nucleic acid when the reaction was initiated. This phenomenon is referred to as the plateau effect, which should be taken into consideration when quantifying the product amplified by PCR.
A technique known as real time PCR is widely employed for target sequence quantification. In this technique, a serial dilution of the target nucleic acid is prepared, each sample is subjected to PCR, and the time course is then taken in real time. The threshold cycle (the Ct value), with which a given amount of amplified product is obtained in a region where amplification exponentially occurs before reaching the level of the plateau effect, is determined. The determined value is plotted on a vertical axis, and the amount of nucleic acid is plotted on a horizontal axis. Thus, a calibration curve is prepared. An unknown sample of interest is subjected to PCR under the same conditions and the Ct value is determined. This enables the quantification of the amount of nucleic acid in the unknown sample. A device for real time detection is generally expensive. If this technique is performed using a common commercial thermal cycler, the sample has to be analyzed each cycle in order to determine the threshold cycle with which a given amount of amplified product is generated. Thus, this technique requires a large amount of labor.
Quantitative competitive PCR is also a widely employed technique. In this technique, a competitor nucleic acid having a sequence similar to that of the target nucleic acid is diluted in a stepwise manner, and the resultants are added to a sample containing the target nucleic acid to be quantified. Depending on the amount of the competitor nucleic acid added, the ratio of the amount of the amplified product from the target nucleic acid to the amount of the amplified product from competitor nucleic acid added, is determined. Accordingly, the point where the amount of the amplified product from target nucleic acid which was added becomes equal to the amount of the amplified product from competitor nucleic acid, represents the amount of the target nucleic acid. Although this technique is relatively simple, the necessity of preparing competitors for each primer complicates the operation. In addition, there is a problem that the amplification efficiency of the target nucleic acid may differ from that of the competitor nucleic acid.
In light of the above, what is needed are relatively simple and inexpensive methods for quantitating nucleic acids, and other targets, in a sample.